This invention relates to methods for preparing and isolating fragments of immunoglobulins from the whole proteins in a rapid and highly efficient manner.
Proteolytic digestion of immunoglobulin molecules have been used for some time to investigate the immunological and other biological properties of antibodies and fragments thereof. Papain and pepsin are the most commonly used proteases to generate F.sub.(ab) and F.sub.(ab')2 fragments, respectively. Both of these fragments retain some immunological reactivity, although only the F.sub.(ab')2 fragment further retains the ability to form precipitable antigen-antibody complexes. The original work of Porter used soluble forms of papain and pepsin; the fragmented antibody and the protease were separated after proteolysis by rather cumbersome ion exchange chromatography [Porter, R. R., Biochem. J. 73:119 (1959)].
While early studies of protease digestion of immunoglobulin G employed soluble papain or pepsin and long incubation times, Putnam et al demonstrated that by increasing the papain to IgG ratio, the time of incubation could be drastically cut down from 16 hours to 1 hour or less [Putnam, W. P. et al, J. Biol. Chem. 237: 717 (1962)]. Difficulties in removal of papain from the cleavage products as well as the possibility of papain contaminating the final F.sub.(ab) preparations, become significantly greater with increased protease concentration. Generally, extensive further purification by non-specific methods such as ion exchange or other chromatographic techniques have heretofore been necessary when elevated protease levels were employed to achieve acceptable reaction rates.
Subsequent to proteolysis, fragments having antigen binding sites [F.sub.(ab), F.sub.(ab')2 ] and the protease may be separated from fragments having no antigen binding site (F.sub.c) and from intact antibody using an immobilized Protein A which specifically binds to the F.sub.c portion of fragmented or intact antibody. The F.sub.c and any undigested IgG may be subsequently eluted from the Protein A column using buffers at acid pH. Removal of the protease has, however, remained a problem.
The development of activated supports has greatly facilitated the preparation of various immobilized proteins and enzymes, including immobilized forms of pepsin and papain which have also been used for the specific fragmentation of antibody. One advantage in using immobilized papain and pepsin is that removal thereof from the reaction mixture may be effected by simple physical separation of the protease subsequent to the digestion of IgG.
A main drawback of this procedure is the slow kinetics of the digestion of IgG by immobilized protease. While the contamination of papain or pepsin could be minimized or eliminated using immobilized protease, the reactions then are in a nonhomogeneous phase, resulting in rates of cleavage which are considerably slower and yields much lower than with systems using homogeneous enzymes. Long incubation times are generally necessary, and incomplete digestion of the immunoglobulin prevents the attainment of adequate yields. It is suspected that limitations on substrate diffusion and steric hindrance inherent in an immobilized enzyme system are probably the major factors causing the reduction in the apparent reaction rate, relative to the kinetics observed with the free enzymes in solution.
It is therefore an object of the present invention to provide methods for fragmentation of immunoglobulins which obviate the problems inherent in the methods heretofore employed for this purpose.
In particular, it is an object of the present invention to provide methods for fragmentation of immunoglobulins, whereby the times required for protease incubation are reduced, the yields of subsequently recovered immunoglobulin fragments are increased, and the separation of IgG and fragments thereof from protease is rapid, simple and effective.